BUSULFAN-GLUTATHIONE CONJUGATION CATALYZED BY HUMAN LIVER CYTOSOLIC GLUTATHIONE S-TRANSFERASES

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human l iver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1 -1. Human liver microsomes and cytosol were incubated with 40 mu M bus ulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfa n tetrahydrothiophenium ion (THT+) in a concentration-dependent manner , whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6,50-7,75), with the ma ximum difference at pH 7.4. Due to the limited aqueous solubility of b usulfan, a K-m for busulfan was not determined, The intrinsic clearanc e (V-max/K-m) of busulfan conjugation was 0.167 mu l/min/mg with 50-12 00 mu M busulfan and 1 mM GSH. GSH V-max and K-m for busulfan conjugat ion were 30.6 pmol/min/mg and 312 mu M, respectively. Ethacrynic acid (0.03-15 mu M) inhibited cytosolic busulfan-conjugating activity with 40 mu M busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decr eased 97% by 15 mu M ethacrynic acid with no effect on the spontaneous reaction, In incubations with affinity-purified liver GST and GST-alp ha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 mu l/min/mg, respectively. Busulfan is a GST substrate with a hig h K-m relative to concentrations achieved clinically (1-8 mu M).